PRP8蛋白质反式剪接系统的建立

A New Protein Trans-splicing System of PRP8 Split Intein

真菌病原体Cryptococcus neoformans AD血清型剪接体蛋白PRP8蛋白质内含子是目前发现的第2个存在于真核生物体核基因组中的蛋白质内含子.它的宿主基因prp8编码的PRP8蛋白作为剪接体的1个组分,是1个高度保守的mRNA剪接蛋白.将组氨酸标签插入克隆自真菌病原体Cryptococcus neoformans AD血清型的PRP8蛋白质内含子中,并将该蛋白质内含子进行人工断裂,获得断裂蛋白质内含子,在大肠杆菌中鉴定其剪接活性.研究结果表明:所获得的改造型蛋白质内含子均表现出高效的剪接活性.利用此Cryptococcus neoformans AD血清型PRP8断裂蛋白质内含子,成功构建了蛋白质反式剪接系统.这一反式剪接系统可用于其他蛋白质的连接与合成,有望成为蛋白质工程中的一种有用工具.

The PRP8 intein from Cryptococcus neoformans is the second intein to be found in a eukaryote nuclear genome. The host gene of PRP8 intein, prp8, encodes a highly conserved mRNA splicing protein found as a part of the spliceosome. In this study, we introduced a His-tag into the PRP8 intein from Cryptococcus neoformans serotype AD, and split this intein artificially. The resulting mini-intein with His-tag and split intein were shown to involved protein splicing when tested in E. coil. Therefore a new protein trans- splicing system using an artificial PRP8 split intein from Cryptococcus neoformans serotype AD was successfully constructed and was shown to be efficient in ligation of flanking proteins. This protein trans-splicing system is a potentially useful tool in protein engineering, such as for protein purification and ligation.